Handbook of Microbiological Media, Fourth Edition part 180 pdf

1.0mg CuSO4·5H2O ...5.0µg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L.. 1.0mg CuSO4·5H2O...5.0μg Preparation of Trace Ele

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Thiosulfate Salts Broth 1785

FeSO4·7H2O 4.99g

CoCl2·6H2O 1.61g

CuSO4·5H2O 1.57g

(NH4)6Mo7O24·4H2O 1.1g

Preparation of Vishniac and Santer Trace Metals: Add

com-ponents to distilled/deionized water and bring volume to 1.0L Adjust

pH to 6.0 with KOH Mix thoroughly

Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L Mix thoroughly Adjust pH to 8.0–8.2

Filter sterilize Aseptically distribute into sterile tubes or flasks

Use: For the cultivation and maintenance of Thiosphaera

pantotro-pha.

Thiosphaera pantotropha Medium

Compositionper 1001.0mL:

Agar 20.0g

Na2HPO4·2H2O 7.9g

KH2PO4 1.5g

NH4Cl 0.3g

MgSO4·7H2O 0.1g

Yeast extract solution 10.0mL

Trace elements solution SL-10 1.0mL

pH 7.5 ± 0.2 at 25°C

Yeast Extract Solution:

Yeast extract 1.0g

Preparation of Yeast Extract Solution: Add yeast extract to

dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly

Autoclave for 15 min at 15 psi pressure–121°C

Trace Elements Solution SL-10:

Compositionper liter:

FeCl2·4H2O 1.5g

CoCl2·6H2O 190.0mg

MnCl2·4H2O 100.0mg

ZnCl2 70.0mg

Na2MoO4·2H2O 36.0mg

NiCl2·6H2O 24.0mg

H3BO3 6.0mg

CuCl2·2H2O 2.0mg

HCl (25% solution) 10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

to 10.0mL of HCl solution Mix thoroughly Add distilled/deionized

water and bring volume to 1.0L Add remaining components Mix

thor-oughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–

121°C

Preparation of Medium: Add components, except yeast extract

so-lution, to distilled/deionized water and bring volume to 990.0mL Mix

thoroughly Gently heat and bring to boiling Autoclave for 15 min at

15 psi pressure–121°C Cool to 50°–55°C Aseptically add 10.0mL of

sterile yeast extract solution Mix thoroughly Pour into sterile Petri

dishes or distribute into sterile tubes

Use: For the cultivation and maintenance of Paracoccus denitrificans.

Thiosulfate Citrate Bile Salts Sucrose Agar

Thiosulfate-Oxidizing Medium

K2HPO4 2.0g MgSO4·7H2O 0.1g CaCl2·2H2O 0.1g FeCl3·6H2O 0.02g (NH4)2SO4 solution 100.0mL Thiosulfate solution 100.0mL

pH 7.8 ± 0.2 at 25°C

(NH 4 ) 2 SO 4 Solution:

(NH4)2SO4 0.1g

Preparation of (NH 4 ) 2 SO 4 Solution: Add the (NH4)2SO4 to dis-tilled/deionized water and bring volume to 100.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

Thiosulfate Solution:

Na2S2O3·5H2O 10.0g

Preparation of Thiosulfate Solution: Add the Na2S2O3·5H2O to distilled/deionized water and bring volume to 100.0mL Mix

thorough-ly Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

Preparation of Medium: Add components, except (NH4)2SO4 so-lution and thiosulfate soso-lution, to distilled/deionized water and bring volume to 800.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C Aseptically add the sterile (NH4)2SO4 solution and the sterile thiosulfate solution Mix

thorough-ly Adjust the pH to 7.8 if necessary Aseptically distribute into sterile tubes or flasks

Use: For the isolation and cultivation of iron and sulfur bacteria

Thiosulfate Salts Broth

Na2S2O3·5H2O 24.81g

NH4·Cl 2.2g

KH2PO4 2.0g Artificial seawater 500.0mL

Artificial Seawater:

NaCl 23.476g MgCl2 4.981g

Na2SO4 3.917g CaCl2 1.102g KCl 0.664g NaHCO3 0.192g KBr 0.096g

H3BO3 0.026g SrCl3 0.024g NaF 3.0mg

pH 5.0 ± 0.2 at 25°C

Preparation of Artificial Seawater: Add components to distilled/ deionized water and bring volume to 1.0L Mix thoroughly

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 5.0 Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure– 121°C

Use: For the cultivation of Thiobacillus species.

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1786 Thiothrix Agar

Thiothrix Agar

(DSMZ Medium 573)

Composition per liter:

Agar 12.0g

NH4Cl 0.2g

Na-acetate 0.1g

K2HPO4 0.01g

MgSO4·7H2O 0.01g

CaSO4 (saturated solution) 20.0mL

Na2S·9H2O solution 10.0mL

Trace elements solution 5.0mL

pH 7.5 ± 0.2 at 25°C

Na 2 S·9H 2 O Solution:

Composition per 10.0mL:

Na2S·9H2O 0.3g

Preparation of Na 2 S·9H 2 O Solution: Add Na2S·9H2O to

dis-tilled/deionized water and bring volume to 10.0mL Sparge with N2

Autoclave for 15 min at 15 psi pressure–121°C Cool to 25°C Store

an-aerobically

Trace Elements Solution:

FeSO4·7H2O 0.7g

EDTA 0.2g

ZnSO4·7H2O 10.0mg

H3BO3 10.0mg

MnSO4·4H2O 2.0mg

Co(NO3)2 1.0mg

Na2MoO4·4H2O 1.0mg

CuSO4·5H2O 5.0µg

Preparation of Trace Elements Solution: Add components to

distilled/deionized water and bring volume to 1.0L Mix thoroughly

Preparation of Medium: Add components, except Na2S·9H2O

so-lution, to distilled/deionized water and bring volume to 990.0mL Mix

thoroughly Gently heat and bring to boiling Autoclave for 15 min at

15 psi pressure–121°C Cool to 50°C Aseptically add 10.0mL sterile

Na2S·9H2O solution Mix thoroughly Pour into Petri dishes or

asepti-cally distribute into sterile tubes

Use: For the cultivation and maintenance of Thiothrix nivea.

Thiothrix Agar

Agar 12.0g

NH4Cl 0.2g

Sodium acetate 0.1g

K2HPO4 0.01g

MgSO4·7H2O 0.01g

CaSO4 (saturated solution) 20.0mL

Trace elements solution 5.0mL

pH 7.5 ± 0.2 at 25°C

Na2S·9H2O 1.0g

Autoclave for 15 min at 15 psi pressure–121°C

FeSO4·7H2O 0.7g EDTA 0.2g ZnSO4·7H2O 0.01g MnSO4·4H2O 0.002g

H3BO3 10.0mg CO(NO3)2 1.0mg

Na2MoO4·2H2O 1.0mg CuSO4·5H2O 5.0μg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly

Preparation of Medium: Add components, except Na2S·9H2O so-lution, to distilled/deionized water and bring volume to 1.0L Mix thor-oughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to 50°–55°C Aseptically add 3.0mL of sterile

Na2S·9H2O solution Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Thiothrix Medium

NH4Cl 0.2g Na-acetate 0.1g

K2HPO4 0.01g MgSO4·7H2O 0.01g CaSO4 (saturated solution) 20.0mL

Na2S·9H2O solution 10.0mL Trace elements solution 5.0mL

pH 7.5 ± 0.2 at 25°C

Na2S·9H2O 0.3g

Preparation of Na 2 S·9H 2 O Solution: Add Na2S·9H2O to dis-tilled/deionized water and bring volume to 10.0mL Sparge with N2 Autoclave for 15 min at 15 psi pressure–121°C Cool to 25°C Store an-aerobically

FeSO4·7H2O 0.7g EDTA 0.2g ZnSO4·7H2O 10.0mg

H3BO3 10.0mg MnSO4·4H2O 2.0mg Co(NO3)2 1.0mg

Na2MoO4·4H2O 1.0mg CuSO4·5H2O 5.0µg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly

Preparation of Medium: Add components, except Na2S·9H2O so-lution, to distilled/deionized water and bring volume to 990.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C

Aseptical-ly add 10.0mL sterile Na2S·9H2O solution Mix thoroughly

Aseptical-ly distribute into sterile tubes or flasks

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Tindallia Medium 1787

Thorne Medium, Modified

Glycerol 20.0g

L-Glutamic acid 4.0g

Citric acid 2.0g

MgSO4·7H2O 1.0g

Ferric ammonium citrate 0.5g

K2HPO4 0.5g

pH 7.4 ± 0.2 at 25°C

water and bring volume to 1.0L Mix thoroughly Adjust pH using

NH4OH (not NaOH) Distribute into tubes or flasks Autoclave for 15

min at 15 psi pressure–121°C

Use: For the cultivation of Bacillus licheniformis.

Thymidine Auxotroph XPS Medium

See: XPS Broth with Thymidine

Tibi Medium

Sucrose 100.0–150.0g

Fig, dried, quartered 1

Lemon wedge (0.5cm segment) 1

Preparation of Medium: Add components to tap water and bring

volume to 1.0L in a 1.0L Erlenmeyer flask fitted with a cotton stopper

Autoclave for 15 min at 15 psi pressure–121°C Cool to room

temper-ature Inoculate with about 50.0mL of Tibi grains

Use: For the cultivation of osmophilic bacteria and fungi from tibi

grains

Tieghemiomyces Medium

Agar 20.0g

Casein hydrolysate 10.0g

KH2PO4 1.0g

MgSO4·7H2O 0.5g

ZnSO4·7H2O 0.2mg

FeSO4 0.2mg

MnSO4·H2O 0.2mg

Thiamine 0.2mg

Biotin 0.01mg

Glycerol 20.0mL

pH 6.0–6.5 at 25°C

water and bring volume to 1.0L Mix thoroughly Gently heat and bring

to boiling Adjust pH to 6.0–6.5 Distribute into tubes or flasks

Auto-clave for 15 min at 15 psi pressure–121°C Pour into sterile Petri dishes

or leave in tubes

Use: For the cultivation and maintenance of Tieghemiomyces

parasit-icus

Tindallia Medium

NaCl 10.0g

Na2CO3 8.0g

Yeast extract 4.0g

NH4Cl 0.5g

KCl 0.2g

K2HPO4 0.2g

Na2S·9H2O solution 10.0mL Trace elements solution SL-10 1.0mL Vitamin solution 1.0mL

pH 9.0 ± 0.2 at 25°C

Vitamin Solution:

Pyridoxine-HCl 10.0mg Thiamine-HCl·2H2O 5.0mg Riboflavin 5.0mg Nicotinic acid 5.0mg

D-Ca-pantothenate 5.0mg

p-Aminobenzoic acid 5.0mg

Lipoic acid 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B12 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L Mix thoroughly Sparge with 80% H2 + 20% CO2 Filter sterilize

Na2S·9H2O 0.5g

Preparation of Na 2 S·9H 2 O Solution: Add Na2S·9H2O to dis-tilled/deionized water and bring volume to 10.0mL Sparge with N2 Autoclave for 15 min at 15 psi pressure–121°C Cool to 25°C Store an-aerobically

Preparation of Medium: Prepare and dispense medium under 100% N2 Add components, except vitamin solution, to distilled/deion-ized water and bring volume to 999.0mL Mix thoroughly Gently heat and bring to boiling Boil for 5 min Cool to 25°C while sparging with 100% N2 Adjust pH to 9.0 Autoclave for 15 min at 15 psi pressure– 121°C Cool to 25°C Aseptically and anaerobically add 1.0mL vita-min solution Mix thoroughly Aseptically and anaerobically under 100% N2 distribute into sterile tubes or bottles

Use: For the cultivation of Tindallia magadiensis.

Tindallia Medium

NaCl 30.0g Peptone 5.0g

NH4Cl 1.0g Yeast extract 0.5g KCl 0.2g

K2HPO4 0.2g MgCl2·6H2O 0.1g Resazurin 1.0mg Carbonate solution 10.0mL Bicarbonate solution 10.0mL Sulfide solution 10.0mL Vitamin solution 2.0mL Trace elements solution 1.0mL

pH 9.7 ± 0.2 at 25°C

Sulfide Solution : Compositionper 10.0mL:

Na2S·9H2O 0.25g

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1788 Tinsdale Agar

Preparation of Sulfide Solution: Add Na2S·9H2O to

distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Autoclave

under 100% N2 for 15 min at 15 psi pressure–121°C Cool to room

temperature

Bicarbonate Solution :

NaHCO3 1.5g

Preparation of Bicarbonate Solution: Add components to

Sparge with a gas mixture of 80% N2 + 20% CO2 Filter sterilize

Carbonate Solution :

Na2CO3 2.76g

Preparation of Carbonate Solution: Add components to

Sparge with a gas mixture of 80% N2 + 20% CO2 Filter sterilize

Pyridoxine-HCl 10.0mg

Thiamine-HCl·2H2O 5.0mg

Riboflavin 5.0mg

Nicotinic acid 5.0mg

Lipoic acid 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Vitamin B12 0.1mg

Preparation of Vitamin Solution: Add components to distilled/

deionized water and bring volume to 1.0L Mix thoroughly Sparge

with 80% H2 + 20% CO2 Filter sterilize

MnCl2·4H2O 0.72g

Fe(NH4)2(SO4)2·6H2O 0.4g

FeSO4·7H2O 0.2g

CoCl2·6H2O 0.2g

ZnSO4·7H2O 0.2g

NiCl2·6H2O 0.1g

Na2MoO4·2H2O 0.02g

CuSO4·5H2O 0.02g

H3BO3 0.02g

KAl(SO4)2·12H2O 0.02g

HCl 5.0mL

Preparation of Trace Elements: Add components to

distilled/de-ionized water and bring volume to 200.0mL Mix thoroughly

Auto-clave for 15 min at 15 psi pressure–121°C

Preparation of Medium: Add components, except sulfide,

bicar-bonate, carbicar-bonate, and vitamin solutions, to distilled/deionized water

and bring volume to 970.0mL Mix thoroughly Sparge with 80% N2 +

20% CO2 gas mixture Dispense into culture vessels Autoclave for 15

min at 15 psi pressure–121°C Cool to room temperature Aseptically

add sulfide, carbonate, bicarbonate, and vitamin solutions Mix

thor-oughly Adjust pH to 9.5.–10.0

Use: For the cultivation of Tindallia spp.

Tinsdale Agar

Proteose peptone 20.0g Agar 15.0g NaCl 5.0g Yeast extract 5.0g L-Cystine 0.24g Tinsdale supplement 150.0mL

pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Di-agnostic Systems and Oxoid Unipath

Tinsdale Supplement:

Na2S2O3 0.43g

K2TeO3 0.35g Serum 100.0mL

Caution: Potassium tellurite is toxic

Preparation of Tinsdale Supplement: Add Na2S2O3 and K2TeO3

to serum Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except Tinsdale sup-plement, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Gently heat and bring to boiling Autoclave for 15 min at

15 psi pressure–121°C Cool to 50°–55°C Aseptically add 100.0mL of sterile Tinsdale supplement Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the primary isolation and identification of Corynebacterium diphtheriae.

Tinsdale HiVeg Agar Base with Tinsdale Supplement

Plant peptone 20.0g Agar 15.0g NaCl 5.0g

Na2S2O3 0.43g

L-Cystine 0.24g Tinsdale supplement 150.0mL

pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-Media

Tinsdale Supplement:

Na2S2O3 0.43g

K2TeO3 0.35g Serum 100.0mL

Caution: Potassium tellurite is toxic

Preparation of Tinsdale Supplement: Add Na2S2O3 and K2TeO3

to serum Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except Tinsdale sup-plement, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Gently heat and bring to boiling Autoclave for 15 min at

15 psi pressure–121°C Cool to 50°–55°C Aseptically add 100.0mL of sterile Tinsdale supplement Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the primary isolation and identification of Corynebacterium diphtheriae.

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Tissue Culture Amino Acids, Minimal Eagle 50X 1789

Tissierella creatinophila Medium

Composition per 1022.0mL:

Creatine 3.8g

Na-formate 2.72g

Yeast extract 2.0g

KCl 1.0g

MgSO4·7H2O 0.5g

NaCl 0.5g

NH4Cl 0.25g

KH2PO4 0.2g

CaCl2·2H2O 0.15g

Resazurin 0.5mg

Na2SeO3·5H2O 0.26mg

NaHCO3 solution 40.0mL

Vitamin solution 10.0mL

Seven vitamin solution 1.0mL

Trace elements solution SL-10 1.0mL

pH 7.5–7.8 ± 0.2 at 25°C

Na 2 S·9H 2 O Solution :

Na2S·9H2O 0.6g

Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C Cool

to room temperature

NaHCO 3 Solution:

NaHCO3 10.0g

Preparation of NaHCO 3 Solution: Add NaHCO3 to

distilled/de-ionized water and bring volume to 100.0mL Mix thoroughly Sparge

with 80% N2 + 20% CO2 Filter sterilize

Pyridoxine-HCl 10.0mg

Riboflavin 5.0mg

Lipoic acid 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Vitamin B12 0.1mg

Preparation of Vitamin Solution: Add components to distilled/

deionized water and bring volume to 1.0L Mix thoroughly Sparge

with 100% N2 Filter sterilize

Seven Vitamin Solution:

Pyridoxine hydrochloride 300.0mg

Vitamin B12 100.0mg

Calcium pantothenate 100.0mg

D(+)-Biotin 20.0mg

Preparation of Seven Vitamin Solution: Add components to dis-tilled/deionized water and bring volume to 1.0L Sparge with 100% N2 Mix thoroughly Filter sterilize

Trace Elements Solution SL-10:

FeCl2·4H2O 1.5g CoCl2·6H2O 190.0mg MnCl2·4H2O 100.0mg ZnCl2 70.0mg

Na2MoO4·2H2O 36.0mg NiCl2·6H2O 24.0mg

H3BO3 6.0mg CuCl2·2H2O 2.0mg HCl (25% solution) 10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

to 10.0mL of HCl solution Mix thoroughly Add distilled/deionized water and bring volume to 1.0L Add remaining components Mix thor-oughly

Preparation of Medium: Prepare and dispense medium under 100%

N2 Add components, except NaHCO3 solution, Na2S·9H2O solution, seven vitamin solution, and vitamin solution, to 950.0mL distilled/de-ionized water Mix thoroughly Sparge with 100% N2 + 20% CO2 Adjust

pH to 7.6 Dispense 10.0mL aliquots into bottles Autoclave for 15 min

at 15 psi pressure–121°C For each 10.0mL medium aseptically and an-aerobically inject from sterile stock solutions 0.4mL NaHCO3 solution, 0.2mL Na2S·9H2O solution, 0.1mL vitamin solution, and 0.01mL

sev-en vitamin solution Final pH should be 7.5–7.8

Use: For the cultivation of Tissierella creatinophila.

Tissue Culture Amino Acids, HeLa 100X (TC Amino Acids, HeLa 100X)

L-Lysine 0.029g L-Isoleucine 0.026g L-Leucine 0.026g L-Threonine 0.023g L-Valine 0.023g L-Tyrosine 0.018g L-Arginine 0.017g L-Phenylalanine 0.016g L-Cystine 0.012g L-Histidine 7.8mg L-Methionine 7.5mg L-Tryptophan 4.1mg

pH 7.2–7.4 at 25°C

Preparation of Tissue Culture Amino Acids, HeLa 100X:

Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.2–7.4 Filter sterilize

Use: For the preparation of Eagle HeLa medium for tissue culture pro-cedures and virus studies

Tissue Culture Amino Acids, Minimal Eagle 50X (TC Amino Acids, Minimal Eagle 50X)

L-Arginine 0.1g L-Lysine 0.058g L-Isoleucine 0.052g L-Leucine 0.052g

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1790 Tissue Culture Dulbecco Solution

L-Threonine 0.048g

L-Valine 0.046g

L-Tyrosine 0.036g

L-Phenylalanine 0.032g

L-Histidine 0.031g

L-Cystine 0.024g

L-Methionine 0.015g

L-Tryptophan 0.01g

pH 7.2–7.4 at 25°C

Preparation of Tissue Culture Amino Acids, Minimal Eagle

50X: Add components to distilled/deionized water and bring volume

to 1.0L Mix thoroughly Adjust pH to 7.2–7.4 Filter sterilize

Use: For the preparation of TC minimal medium Eagle for tissue

cul-ture procedures and virus studies

Tissue Culture Dulbecco Solution

(TC Dulbecco Solution)

NaCl 8.0g

Na2HPO4 1.15g

KH2PO4 0.2g

KCl 0.2g

CaCl2·2H2O 0.1g

MgCl2·6H2O 0.1g

pH 7.2–7.4 at 25°C

Preparation of Tissue Culture Dulbecco Solution: Add

com-ponents to distilled/deionized water and bring volume to 1.0L Mix

thoroughly Adjust pH to 7.2–7.4 Filter sterilize

Use: For use in tissue culture and virus preparations

Tissue Culture Earle Solution

(TC Earle Solution)

NaCl 6.8g

NaHCO3 2.2g

Glucose 1.0g

KCl 0.4g

CaCl2·2H2O 0.2g

NaH2PO4 0.125g

MgSO4·7H2O 0.1g

Phenol Red (1% solution) 2.0mL

pH 7.2–7.4 at 25°C

Preparation of Tissue Culture Earle Solution: Add

compo-nents, except Phenol Red, to distilled/deionized water and bring

vol-ume to 1.0L Mix thoroughly Add 2.0mL of Phenol Red solution

Adjust pH to 7.2–7.4 Filter sterilize

Use: For use in tissue culture and virus preparations

Tissue Culture Hanks Solution

(TC Hanks Solution)

NaCl 8.0g

Glucose 1.0g

KCl 0.4g

NaHCO3 0.35g

CaCl2·2H2O 0.14g

MgCl2·6H2O 0.1g

MgSO4·7H2O 0.1g

Na2HPO4 0.06g

KH2PO4 0.06g Phenol Red 0.02g

pH 7.2–7.4 at 25°C

Source: This medium is available as a premixed solution from BD Di-agnostic Systems

Preparation of Tissue Culture Hanks Solution: Add compo-nents to distilled/deionized water and bring volume to 1.0L Mix thor-oughly Adjust pH to 7.2–7.4 Filter sterilize

Use: For use in tissue culture procedures

Tissue Culture Medium 199 (TC Medium 199)

NaCl 8.0g Glucose 1.0g KCl 0.4g NaHCO3 0.35g DL-Glutamic acid 0.15g CaCl2·2H2O 0.14g DL-Leucine 0.12g L-Glutamine 0.1g MgSO4·7H2O 0.1g L-Arginine 0.07g L-Lysine 0.07g DL-Aspartic acid 0.06g

Na2HPO4 0.06g

KH2PO4 0.06g DL-Threonine 0.06g DL-Alanine 0.05g Glycine 0.05g DL-Phenylalanine 0.05g DL-Serine 0.05g Sodium acetate 0.05g DL-Valine 0.05g DL-Isoleucine 0.04g L-Proline 0.04g L-Tyrosine 0.04g DL-Methionine 0.03g L-Cystine 0.02g L-Histidine 0.02g Phenol Red 0.02g DL-Tryptophan 0.02g Adenine 0.01g L-Hydroxyproline 0.01g Tween™ 80 5.0mg Adenosine triphosphate 1.0mg Choline 0.5mg Deoxyribose 0.5mg Ribose 0.5mg Guanine 0.3mg Hypoxanthine 0.3mg Thymine 0.3mg Uracil 0.3mg Xanthine 0.3mg Adenylic acid 0.2mg Cholesterol 0.2mg Calciferol 0.1mg Fe(NO3)3·9H2O 0.1mg L-Cysteine 0.1mg

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1792 Tissue Culture Medium Eagle, HeLa

Glutamine Solution:

L-Glutamine 5.0g

NaCl (0.85% solution) 100.0mL

Preparation of Glutamine Solution: Add the glutamine to the

0.85% NaCl solution Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except glutamine and

serum, to distilled/deionized water and bring volume to 1.0L Mix

thor-oughly Adjust pH to 7.2–7.4 Filter sterilize Aseptically add 6.0mL of

sterile glutamine solution and 50.0–100.0mL of sterile serum Human

serum, bovine serum, horse serum, or fetal calf serum may be used Mix

thoroughly

Use: For use as a base in the preparation of liquid media used for the

cultivation of tissue culture cell lines

Tissue Culture Medium Eagle, HeLa

(TC Medium Eagle, HeLa)

NaCl 5.85g

NaHCO3 1.68g

Glucose 0.9g

KCl 0.373g

NaH2PO4 0.138g

MgCl2·6H2O 0.12g

CaCl2·2H2O 0.11g

L-Lysine 0.0269g

L-Isoleucine 0.0262g

L-Leucine 0.0262g

L-Threonine 0.0238g

L-Valine 0.0234g

L-Tyrosine 0.0181g

L-Arginine 0.0174g

L-Cystine 0.012g

L-Histidine 7.8mg

L-Methionine 7.5mg

Phenol Red 5.0mg

L-Tryptophan 4.1mg

Folic acid 0.44mg

Thiamine·HCl 0.34mg

Biotin 0.24mg

Pantothenic acid 0.22mg

Pyridoxal·HCl 0.2mg

Choline chloride 0.14mg

Nicotinamide 0.12mg

Riboflavin 0.04mg

Serum 50.0mL–100.0mL

Glutamine solution 6.0mL

pH 7.2–7.4 at 25°C

Glutamine Solution:

L-Glutamine 5.0g

NaCl (0.85% solution) 100.0mL

Preparation of Glutamine Solution: Add the glutamine to the

0.85% NaCl solution Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except glutamine and

serum, to distilled/deionized water and bring volume to 1.0L Mix

thor-oughly Adjust pH to 7.2–7.4 Filter sterilize Aseptically add 6.0mL of

sterile glutamine solution and 50.0–100.0mL of sterile serum Human

serum, bovine serum, horse serum, or fetal calf serum may be used Mix thoroughly

Use: For the cultivation and maintenance of HeLa and other cell lines

in tissue culture, and for studying the cytopathogenicity of viral agents

Tissue Culture Medium Ham F10 (TC Medium Ham F10)

NaCl 7.4g Glucose 1.1g

Na2HPO4 0.29g KCl 0.285g L-Arginine 0.211g MgSO4·7H2O 0.153g L-Glutamine 0.1462g Sodium pyruvate 0.11g

KH2PO4 0.083g CaCl2·2H2O 0.044g L-Cystine 0.0315g L-Lysine 0.0293g L-Histidine 0.021g L-Asparagine 0.015g L-Glutamic acid 0.0147g L-Aspartic acid 0.0133g L-Leucine 0.0131g L-Proline 0.0115g L-Serine 0.0105g L-Alanine 8.91mg Glycine 7.51mg L-Phenylalanine 4.96mg L-Methionine 4.48mg Hypoxanthine 4.0mg L-Threonine 3.57mg L-Valine 3.5mg L-Isoleucine 2.6mg L-Tyrosine 1.81mg Cyanocobalamin 1.3mg Folic acid 1.3mg Phenol Red 1.2mg Thiamine·HCl 1.0mg FeSO4·7H2O 0.83mg Calcium pantothenate 0.7mg Thymidine 0.7mg Choline chloride 0.69mg Niacinamide 0.6mg L-Tryptophan 0.6mg

i-Inositol 0.54mg

Riboflavin 0.37mg Lipoic acid 0.2mg Pyridoxine·HCl 0.2mg ZnSO4·7H2O 0.028mg Biotin 0.024mg CuSO4·5H2O 2.5μg Fetal calf serum 50.0–100.0mL

pH 7.2–7.4 at 25°C

Preparation of Medium: Add components, except fetal calf serum,

to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.2–7.4 with 10% Na2CO3 solution Filter sterilize Asep-tically add 50.0–100.0mL of sterile fetal calf serum Mix thoroughly

Use: For the cultivation of a wide variety of cell lines in tissue culture

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Tissue Culture Medium RPMI No 1640 1793

Tissue Culture Medium NCTC 109

(TC Medium NCTC 109)

NaCl 6.8g

NaHCO3 2.2g

Glucose 1.0g

KCl 0.4g

L-Cysteine 0.26g

CaCl2·2H2O 0.2g

NaH2PO4 0.14g

L-Glutamine 0.14g

MgSO4·7H2O 0.1g

Sodium acetate 0.05g

Ascorbic acid 0.05g

L-Alanine 0.03g

L-Lysine 0.03g

L-Arginine 0.026g

L-Valine 0.025g

L-Leucine 0.02g

Phenol Red 0.02g

L-Histidine 0.019g

L-Threonine 0.019g

L-Isoleucine 0.018g

L-Tryptophan 0.017.g

L-Tyrosine 0.016g

Glycine 0.014g

Tween™ 80 0.012g

L-Serine 0.011g

L-Cystine 0.01g

Glutathione 0.01g

Cyanocobalamin 0.01g

Deoxycytidine 0.01g

Deoxyguanosine 0.01g

Deoxyadenosine 0.01g

Thymidine 0.01g

L-Aspartic acid 9.91mg

L-Glutamic acid 8.26mg

L-Arginine 8.09mg

L-Ornithine 7.38mg

Nicotinamide adenine dinucleotide 7.0mg

L-Proline 6.13mg

L-α-N-butyric acid 5.51mg

L-Methionine 4.44mg

L-Taurine 4.18mg

L-Hydroxyproline 4.09mg

D-Glucosamine 3.2mg

Coenzyme A 2.5mg

Glucuronolactone 1.8mg

Sodium glucuronate 1.8mg

Choline chloride 1.25mg

Cocarboxylase 1.0mg

Flavin adenine dinucleotide 1.0mg

Uridine triphosphate 1.0mg

Nicotinamide adenine

dinucleotide phosphate 1.0mg

Vitamin A 0.25mg

Calciferol 0.25mg

i-Inositol 0.125mg

5-Methylcytosine 0.1mg

Pyridoxine·HCl 0.0625mg

Pyridoxal·HCl 0.0625mg Niacin 0.0625mg Niacinamide 0.0625mg Biotin 0.025mg Folic acid 0.025mg Menadione 0.025mg Pantothenate 0.025mg Riboflavin 0.025mg Thiamine·HCl 0.025mg α-Tocopherol phosphate 0.025mg Serum 50.0–100.0mL

pH 7.2–7.4 at 25°C

Preparation of Medium: Add components, except serum, to dis-tilled/deionized water and bring volume to 1.0L Mix thoroughly Ad-just pH to 7.2–7.4 with 10% Na2CO3 solution Filter sterilize Asepti-cally add 50.0–100.0mL of sterile serum Human serum, bovine serum, horse serum, or fetal calf serum may be used Mix thoroughly

Tissue Culture Medium RPMI No 1640 (TC Medium RPMI #1640)

NaCl 6.46g Glucose 2.0g NaHCO3 2.0g NaH2PO4 1.512g KCl 0.4g L-Glutamine 0.3g L-Arginine 0.2g Calcium nitrate 0.1g MgSO4·7H2O 0.1g L-Asparagine 0.05g L-Cystine 0.05g L-Isoleucine 0.05g L-Leucine 0.05g L-Lysine·HCl 0.04g Inositol 0.035g L-Serine 0.03g Hydroxy-L-proline 0.02g L-Aspartic acid 0.02g L-Glutamic acid 0.02g L-Proline 0.02g L-Threonine 0.02g L-Tyrosine 0.02g L-Valine 0.02g L-Histidine 0.015g L-Methionine 0.015g L-Phenylalanine 0.015g Glycine 0.01g L-Tryptophan 5.0mg Phenol Red 5.0mg Choline chloride 3.0mg

Folic acid 1.0mg Glutathione 1.0mg Nicotinamide 1.0mg Pyridoxine·HCl 1.0mg Thiamine·HCl 1.0mg Calcium pantothenate 0.25mg Biotin 0.2mg

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1794 Tissue Culture Minimal Medium Eagle

Riboflavin 0.2mg

Vitamin B12 5.0mg

Serum 50.0–100.0mL

pH 7.2–7.4 at 25°C

Source: This medium is available as a premixed powder and solution

from BD Diagnostic Systems

Preparation of Medium: Add components, except serum, to

dis-tilled/deionized water and bring volume to 1.0L Mix thoroughly

Ad-just pH to 7.2–7.4 with 10% Na2CO3 solution Filter sterilize

Asepti-cally add 50.0–100.0mL of sterile serum Human serum, bovine serum,

horse serum, or fetal calf serum may be used Mix thoroughly

Tissue Culture Minimal Medium Eagle

Sterile salt solution 944.0mL

TC amino acids, minimal Eagle 50X 20.0mL

TC NaHCO3, 10% 20.0mL

TC vitamins, minimal Eagle 100X 10.0mL

TC glutamine, 5% 6.0mL

pH 7.2–7.4 at 25°C

Sterile Salt Solution:

NaCl 6.8g

Glucose 1.0g

KCl 0.4g

CaCl2 0.2g

MgCl2 0.2g

NaH2PO4 0.15g

Preparation of Sterile Salt Solution: Add components to

Filter sterilize

TC Amino Acids, Minimal Eagle 50X:

L-Arginine 0.1g

L-Lysine 0.06g

L-Isoleucine 0.05g

L-Leucine 0.05g

L-Threonine 0.05g

L-Valine 0.05g

L-Tyrosine 0.04g

L-Histidine 0.03g

L-Cystine 0.02g

L-Methionine 0.02g

L-Tryptophan 0.01g

Preparation of TC Amino Acids, Minimal Eagle 50X: Add

components to distilled/deionized water and bring volume to 1.0L Mix

thoroughly Adjust pH to 7.2–7.4 Filter sterilize

NaHCO3 10.0g

Preparation of TC NaHCO 3 , 10%: Add NaHCO3 to

distilled/de-ionized water and bring volume to 100.0mL Mix thoroughly Filter

sterilize

TC Vitamins, Minimal Eagle 100X:

Inositol 2.0mg Calcium pantothenate 1.0mg Choline chloride 1.0mg Folic acid 1.0mg Nicotinamide 1.0mg Pyridoxal 1.0mg Thiamine·HCl 1.0mg Riboflavin 0.1mg

Preparation of TC Vitamins, Minimal Eagle 100X : Add

com-ponents to distilled/deionized water and bring volume to 1.0L Mix thor-oughly Filter sterilize

TC Glutamine, 5%:

L-Glutamine 5.0g NaCl (0.85% solution) 100.0mL

Preparation of TC Glutamine, 5%: Add the glutamine to the 0.85% NaCl solution Mix thoroughly Filter sterilize

Preparation of Medium: Aseptically combine 944.0mL of sterile salt solution, 20.0mL of sterile TC amino acids, minimal Eagle 50X, 20.0mL of sterile TC NaHCO3, 10%, 10.0mL of sterile TC vitamins, minimal Eagle 100X, and 6.0mL of sterile TC glutamine, 5% Mix thoroughly Adjust pH to 7.2–7.4 if necessary

Use: For the cultivation of mammalian cells in monolayer or suspen-sion for tissue culture procedures and virus preparation

Tissue Culture Minimal Medium Eagle with Earle Balanced Salts Solution (TC Minimal Medium Eagle with Earle BSS)

NaCl 6.8g Glucose 1.0g KCl 0.4g CaCl2·2H2O 0.2g MgCl2·6H2O 0.2g NaH2PO4 0.15g L-Arginine 0.1g L-Lysine 0.06g L-Isoleucine 0.05g L-Leucine 0.05g L-Threonine 0.05g L-Valine 0.05g L-Tyrosine 0.04g L-Phenylalanine 0.03g L-Histidine 0.03g L-Cystine 0.02g L-Methionine 0.02g L-Tryptophan 0.01g

i-Inositol 2.0mg

Calcium pantothenate 1.0mg Choline chloride 1.0mg Folic acid 1.0mg Nicotinamide 1.0mg Pyridoxal 1.0mg Thiamine·HCl 1.0mg Riboflavin 0.1mg Serum 50.0–100.0mL

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